Incubate the hPSC cultures (on MEF feeders) in hPSC medium containing 10 µM Y-27632 (1:1000 dilution of stock) for at least two hours (overnight works as well) before electroporation. Two to three near confluent 6-well plates should provide sufficient cells for each individual experiment (higher than 10X10⁶ hPSCs/experiment)

For a detailed protocol on hPSC cultures on MEF feeders, refer to the collection "Maintenance and inactivation of mouse embryonic fibroblasts (MEFs) as feeder cells for human pluripotent stem cell culture;" dx.doi.org/10.17504/protocols.io.b4pbqvin

hPSCs medium

A	B
DMEM/F12	385 ml
Fetal Bovine Serum (FBS)	75 ml
Knockout Serum Replacement	25 ml
L-Glutamine (100X)	5 ml
Penicillin & Streptomycin (100X)	5 ml
MEM Non-Essential Amino Acids (100X)	5 ml
2-Mercaptoethanol (10,000X)	50 µl
Heat Stable Recombinant Human FGF2 (25µg/ml)*	80 µl

*While we prefer Heat Stable Recombinant Human FGF2, we also have used regular FGF2. Final volume: 500ml

L-Glutamine (100X)

A	B
L-Glutamine, powder	14.6 g
MilliQ H2O	500 ml

2-Mercaptoethanol (10,000X)

A	B
2-Mercaptoethanol	0.78 ml
MilliQ H2O	9.22 ml

Heat Stable Recombinant Human FGF2 (25µg/ml)

A	B
Heat Stable Recombinant Human FGF2	500 µg
0.1% BSA	20 ml

Final volume: 20ml

Y-27632 (1,000X)

A	B
Y-27632	5 mg
DMSO	1.56 ml

hPSCs medium + Rock inhibitor

A	B
hPSCs medium	500 ml
Y-27632 (1,000X)	500 µl

Final volume: 500ml

Wash the hPSC plates twice with DPBS

Add 1 ml Trypsin/well of a 6-well plate and incubate for 0h 5m 0s at 37°C until colonies start detaching from MEF feeders.

Add 2 ml hPSC medium to inactivate the trypsin.

Collect single cell or small clump solution by trituration with P1000 pipette tips into 50 ml conical tube.

Gravity precipitate cell solution for 0h 5m 0s min and remove large clumps of feeders with 5 ml pipette from the bottom of the conical tube.

Centrifuge at225x g

Remove the supernatant and re-suspend the cells at high concentration (higher than 10X10⁶cells/700 µl) in ice-cold DPBS.

Thaw plasmids On ice

Dependent on the specific experiment (ZNFs-, TALEN- or CRISPR/Cas9-based genome editing) we recommend keeping the overall amount of plasmid DNA for each experiment below 50 µg.

For CRISPR/Cas9 genome editing , use:

16 µg pX330-GFP gRNA (alternative: pX330-mcherry/BFP/YFP)

34 µg ssODN (single strand oligonucleotide containing modification, \~100 bp)

For TALEN genome editing , use

7.5 µg for each (left and right) TALEN-nuclease plasmid

26 µg ssODN (single strand oligonucleotide containing modification, \~100 bp)

10 µg pEGFP-N1 (or comparable fluorescent protein expression vector)

For ZFN genome editing , use

7.5 µg for each (left and right) ZFN-nuclease plasmid

26 µg ssODN (single strand oligonucleotide containing modification, \~100 bp)

10 µg pEGFP-N1 (or comparable fluorescent protein expression vector)

For prime editing PE2 strategy, use

33 µg pCMV-PE2-GFP

12 µg pU6-pegRNA

For prime editing PE3 strategy , use

33 µg pCMV-PE2-GFP

12 µg pU6-pegRNA

5 µg pBPK1520-ngRNA

Pipet the proper amount of each plasmid into a micro centrifuge tube and keep On ice (4°C) until electroporation.

Mix cell suspension (higher than 10X10⁶cells/700 µl DPBS) with pre-made transfection solution (plasmid DNA), transfer in Biorad electroporation cuvette (4 mm) and incubate on ice for 0h 10m 0s

Resuspend cell suspension cuvettes (either by careful vortexing or by hand) and pulse cuvettes using Biorad electroporator (Gene Pulser Xcell System, Bio-Rad: 250 V, 500 µF, 4 mm cuvettes).

Carefully collect the cells and transfer to pre-warmed hPSC medium containing 10 µM Y-27632 (1:1000 dilution of stock) and distribute electroporated cell solution on an entire 6-well MEF feeder plate (DR4-MEFs for selection-based experiments and ICR-MEFs for subsequent FACS sorting)

After plating shake the plate/s gently in the incubator and let it sit (37°C; 5% CO₂; 5% O₂).

Refresh hPSC medium daily until FACS or drug selection (usually after 48 – 72 hours).